Gene therapy is a powerful tool that is being used to combat inherited and acquired disease. The ability to introduce transgenes into a cell and restore normal physiology holds the future for ameliorating a variety of inherited and acquired errors in metabolism. Currently, there are roughly 1800 ongoing clinical trials where the transgene is constitutively expressed, using either ubiquitous or tissue-specific promoters. However, for most inherited or acquired disorders the transgene must be tightly regulated and/or titrated with disease progression. An additional major concern is that constitutive transgene expression can be toxic, producing undesirable side effects and even death. For safe and effective protein delivery, the transgene must be appropriately regulated.
The field of gene therapy has seen many significant advances over the past decade3. Viral vectors are available that can effectively express heterologous genes in vivo and provide long-term gene expression to target tissues with minimal toxicity and immune response. One of the most successful applications of this technology has been the restoration of vision for individuals with the retinal degenerative disorder described as Leber's congenital amaurosis (LCA)4,5. Currently there are approximately 1800 ongoing human gene therapy clinical trials that use similar gene replacement therapies; however, for many pathological conditions gene augmentation therapies need to be regulated1. Indeed, gene regulation remains one of the most important and unresolved obstacles for safe and effective development of clinical gene therapeutics.
Most transgene regulatory systems are based upon the classical bacterial operons, where a regulating protein is constitutively produced by one promoter to modulate transcription of a second promoter expressing a functional gene6. A variety of such inducible regulatory systems have been developed utilizing a number of different regulatory proteins. These regulatory proteins are allosterically controlled by effector molecules such as the antibiotic tetracycline (Tet), steroid hormones (ecdysone), anti-steroid hormone analogs (mifepristone and tamoxifen), and immunosuppressant (rapamycin)6. Regardless of how the regulator is controlled, the fundamental problems that have plagued all of these regulatory systems is that effector molecules can produce unwanted side effects, the regulatory circuitry exhibits a high basal level of gene expression with only a modest dynamic range, and many of these systems are too large to easily fit within the packaging constraints of a single viral vector such as a recombinant adeno-associated virus (rAAV). Additionally, the level of regulator protein produced in these systems is constant and dependent on a number of extrinsic variables, such that the system will behave differently in different environments. The resulting switch must be empirically tuned for each particular application to ensure sufficient dynamic range and to minimize leakiness of transgene expression in the uninduced state.
As a consequence there is tremendous interest in developing expression systems in which the dosages of therapeutic transgenes are readily and easily regulated. Transgene regulation has the potential to modulate, stop, and/or resume transgene expression in response to disease evolution. A number of inducible systems have been developed to control transgene expression in mammalian cells.
For example, one inducible activation system for regulating transgene expression in eukaryotic cells was created by fusing the Tet repressor with an activation domain, VP16, from Herpes Simplex Virus. This fusion protein binds to the tetracycline response elements (TREs) located within an inducible promoter, activating transcription either in the presence or the absence of inducer. The two systems, “Tet-On” and “Tet-Off’ both activate expression but respond to doxycycline (Dox) differently; Tet-Off activates expression in the absence of Dox, whereas Tet-On activates in the presence of Dox. Another commonly used switch to regulate transcription relies on rapamycin, an immunosuppressant. Analogous to the Tet-On system, rapamycin mediates heterodimer formation between two proteins; FKBP and FRB; where FKBP is fused to a zinc finger homeodomain and FRB to an activation domain. Transcription is activated by recruiting the activation domain to the promoter that is mediated by rapamycin facilitated dimerization of FKBP and FRP.
These known inducible systems require delivery of multiple components, thereby increasing the potential for immunologic complications. For both tetracycline and rapamycin, repeated administration can lead to toxic (and sometimes life-threatening) side effects. Existing inducible systems also suffer wide variations in expression levels that increase the risk of adverse effects in the host organism and confound scientific experimentation.